To activate and study the Wnt pathway, a wide range of materials and information has been used. Various model organisms explained below are used because of differing developmental characteristics associated with the organisms. Because frogs and mice are exemplary of the organisms of study, they are explained in greater detail below. As will be seen, frogs and mice were used in many of the Examples contained herein. Additionally, various genes and the Wnt pathway are explained.
Background of the Frog.
Frogs, in particular Xenopus, are excellent model organisms for testing embryonic development. Two species of Xenopus are commonly used for testing, Xenopus laevis and Xenopus tropicalis. Both Xenopus species are natives of Africa. Xenopus laevis has been used for many years to investigate the early period of embryonic development. Embryos develop rapidly after fertilization; and a tadpole with a fully functional set of organs forms within a couple of days. Thus, experiments can be conducted on the embryos directly following fertilization. The embryos can develop in a simple saline solution over a few days. The tadpoles are then examined to determine if the experimental intervention had any observable effect. The role of genes in development can be assayed by injecting a tiny amount of any messenger RNA (mRNA) encoding the gene of interest into an early embryo, then once again allowing the embryo to grow into a tadpole.
The Xenopus embryo has long served as a major model for the study of embryonic development because of its numerous advantages, including external development, large size, identifiable blastomeres, and its ability to withstand extensive surgical intervention and culture in vitro. These advantages enable extensive investigation of the earliest embryonic patterning events. In fact, much of the current understanding of early embryonic development derives from experiments performed in the Xenopus embryo.
More particular to the frog, the earliest events of all animal embryos are controlled by mRNAs that are deposited in the egg by the mother. These maternal mRNAs control the embryonic processes that occur prior to the transcription of the embryonic genome. These processes can best be examined in Xenopus because, in these embryos, they occur during an especially long period of time, and because they occur while the embryo is developing externally. Such features have resulted in a detailed cellular and molecular understanding of early patterning events, including a comprehensive view of the role of specific extracellular growth factors, cell surface receptors, and intracellular signaling pathway components. These events include the patterning of the basic body plan, the determination of cell fate, and the early patterning of major organs, including the digestive system, circulatory system, and nervous system. In addition, many of the factors originally identified in Xenopus have been subsequently shown to control numerous later developmental events, as well as other critical biological processes, and oncogenesis. Finally, Xenopus is a major contributor to understanding cell biological and biochemical processes, including chromosome replication; chromatin, cytoskeleton, and nuclear assembly; cell cycle progression; and, intracellular signaling. Thus, Xenopus is ideally suited for studying early embryonic patterning and cell fate determination, later development, and organogenesis, oncogenesis, and cell biological and biochemical processes.
Background of A-P Patterning.
The mechanisms that generate regional differences along the anterior posterior (A-P) axis of the vertebrate nervous system play an important role in pattern formation during development. The classical activation/transformation model proposed by Nieuwkoop suggests that an initial signal induces neural tissue of anterior type and then a second transforming signal differentially acts on it to convert cells to a more posterior character (Nieuwkoop, 1952; Slack and Tannahill, 1992). This transformer or “posteriorizing factor(s)” thus modifies a ground state to generate the full spectrum of neural structures along the A-P axis. However, patterning of the anterior region is clearly more complicated than a simple default state of neural induction. This is highlighted by the presence of local inductive centers, such as the anterior visceral endoderm and the isthmus, which are essential for anterior neural development. Hence, models for a coordinated mechanism of A-P patterning in the nervous system need to integrate the influence of local signals on rostral brain patterning, with the influence of posteriorizing factors that work more generally on the hindbrain and spinal cord.
Analysis of posteriorizing signals in neural patterning is complicated by the tissue interactions and dynamic morphogenetic movements which occur during gastrulation. Xenopus animal caps provide a simplified system for studying patterning events separately from morphogenetic movements. Animal caps alone form epidermis in culture, but when treated with antagonists of Bone Morphogenic Protein (BMP) signaling, such as Noggin, Chordin, Follistatin, or truncated BMP receptors, they adopt an anterior neural fate. Using these molecules as neural inducers, experimental studies in animal caps have provided evidence that fibroblast growth factor (FGF), retinoic acid (RA), and Wnt (Wingless and iNT-1) signaling pathways influence A-P patterning by inducing posterior characters. Wnt is also known as the canonical Wnt pathway and the Wnt planar polarity pathway. Thus, Xenopus embryo assays and experiments in other vertebrates provide more evidence that RA, FGF, and Wnt pathways influence A-P patterning. It is desired to better understand the relative roles of these biochemical cascades, the degree to which they are used at any particular axial level, and how they are integrated in organizing normal A-P patterning.
Mesoderm plays an important early role in A-P patterning of neural tissue. Mesoderm is the middle layer of embryonic cells between the ectoderm and endoderm in triploblastic animals, and forms muscle, connective tissue, blood, lymphoid tissue, the linings of all the body cavities, the serosa of the viscera, the mesenteries, and the epithelia of the blood vessels, lymphatics, kidney, ureter, gonads, genital ducts, and suprarenal cortex. Experiments in Xenopus have shown that planar signals within the neuroectoderm and vertical signals from the underlying mesoderm work in concert to control regional identity of the nervous system. While early A-P specification of the nervous system occurs during gastrulation, it is not irreversibly committed to a particular identity. Grafting experiments in several species reveal plasticity in regional character and show that mesoderm is still playing a role at later stages. For example, analysis of the Hoxb4 gene has shown that its expression pattern is established through interactive signaling between the neural tube and the surrounding mesoderm. Furthermore, somites and paraxial mesoderm are sufficient to re-program Hox expression in the neural tube to a more posterior character when grafted ectopically. The ability of mesoderm to regulate regional character from early gastrula stages and to program motor neuron sub-type identities further emphasizes the importance of mesoderm and its signaling in patterning the developing nervous system.
The study of A-P patterning and focus on the mesoderm is of particular importance in the present invention because such patterning impacts bone development in an embryo. Pathways which control A-P patterning often impact bone development.
As such, it is desired to better understand the process of posteriorization. The identification of new factors that can modulate existing pathways, such as Wnts, FGF, and RA, or which represent novel signaling inputs will be beneficial to understanding how A-P patterning is coordinated. In particular, it is desired to understand how the Wnt pathway is activated and controlled. Xenopus has been used to study A-P patterning, that, in turn, is apparently impacted by the Wnt pathway. Xenopus can also be used to study activators or inhibitors of the Wnt pathway.
Background of Mouse Model.
Mice are also excellent model organisms for testing embryonic development. Mice and humans possess similar genes, mice show many clinical symptoms of human disease, and powerful techniques are available for genetic alterations of the mouse genome. All of these factors make mice excellent experimental models for testing new therapies. Mice share many fundamental biological processes with humans therefore, mice are considered to be one of the most significant laboratory models for human disease and genetic mutations. Research regarding human biological processes and genetic diseases can be greatly enhanced by studying the mouse model for similar biological processes and diseases.
Mice have been a preferred experimental model for a number of years due to their small size, short life span, and the female's ability to produce a litter within two months after her birth. These factors allow researchers to follow a given disease process from beginning to end within a short time frame. For these various reasons, mouse models are preferred for testing new drug therapies, designing novel therapies, and studying genetic diseases potentially also affecting humans.
Genes can be inserted into a fertilized mouse egg by several methods including physical injection. The gene is first attached to a promoter and then is injected into the fertilized egg. The fertilized egg is implanted into a female mouse and the embryo is allowed to develop to a specified given stage for study. Once embryos reach the desired stage of development, they can be harvested and tested to determine experimental results. Alternatively, embryos can be allowed to develop into full-term pups prior to being harvested to determine the results of the experiment.
Because mice are phylogenetically closely related to humans with regards to biological processes and diseases, and because of the rapidity of mouse embryological development, they are considered to be an excellent animal model for the study of human development, biological processes, and disease.
Background of Wnt.
Wnt proteins form a family of highly conserved secreted signaling molecules that regulate cell-to-cell interactions during embryogenesis. Wnt genes and Wnt signaling are also implicated in aberrant cancer cell regulation. Insights into the mechanisms of Wnt action have emerged from several systems: genetics in Drosophila and Caenorhabditis elegans (C. elegans); and, biochemistry in cell culture and ectopic gene expression in Xenopus embryos. Many Wnt genes in the mouse have been mutated, leading to very specific developmental defects. As currently understood, Wnt proteins bind to receptors of the Frizzled family on the cell surface. Through several cytoplasmic relay components, the signal is transduced to β-catenin, which then enters the nucleus and forms a complex with TCF or LEF to activate transcription of Wnt target genes. The extracellular Wnt ligand binds the transmembrane receptor Frizzled (Fz), which activates the cytoplasmic phosphoprotein Dishevelled (Dsh). Activated Dsh inhibits GSK3 β-mediated degradation of β-catenin. β-catenin protein, therefore, accumulates and, in association with transcription factors (TCF-3, TCF-4, LEF), regulates gene transcription in the cell nucleus.
Wnt-proteins, secreted glycoproteins, serve as important signaling molecules during development of invertebrates and vertebrates. They have been shown to play crucial roles in such diverse processes as cancer, organogenesis, and pattern formation. To date, 19 Wnt genes have been isolated in higher vertebrates, 7 have been found in the genome of Drosophila, and 5 in the C. elegans genome. Wnt genes are defined by their sequence similarity to the founding members, Wnt-1 in the mouse (originally called iNT-1) and wingless (Wg) in Drosophila. The genetic analysis of the Wg signaling pathway in Drosophila has led to the identification of many downstream components, which have been shown to be functionally conserved in other organisms. Wg/Wnt-proteins are thought to signal through seven-transmembrane receptors encoded by the Frizzled (Fz) gene family to regulate the stability of an effector protein known as armadillo (Arm) in flies or β-catenin (β-cat) in vertebrates, which eventually leads to the activation of target genes through a complex of Arm/β-cat, with DNA-binding transcription factors of the TCF/LEF family. This pathway is referred to as the canonical Wnt-pathway.
In recent years, evidence has been provided that Wnt signaling in the chick is involved in a variety of processes associated with skeletogenesis, such as chondrogenesis and joint development. Previously, it has been shown that there are at least three Wnt genes, Wnt-4, Wnt-5a, and Wnt-5b, as well as components of the canonical Wnt signaling pathway, expressed in chondrogenic regions, and that there is a fourth Wnt gene, Wnt-14, which is expressed early in the joint forming region (FIG. 1D). Wnt-4 is also expressed in regions of the joint, however, its expression is restricted to cells in the periphery of the joint interzone (FIG. 1C). Wnt-5a expression is restricted to cells in a region of the perichondrium which will develop into the periosteum (FIG. 1A), while the closely related Wnt-5b gene is expressed in a sub-population of prehypertrophic chondrocytes, as well as cells of the outer layer of the perichondrium (FIG. 1B).
Much of what is known about the functional role of Wnt signaling in early vertebrate development comes from experiments with Xenopus. Maternally encoded components of the canonical Wnt signaling pathway function to establish the endogenous dorsal axis. The sperm fertilizing the egg triggers cortical rotation. Vesicles are moved towards the future dorsal side. A dorsal determinant, which is likely to be Dishevelled, is transported with these vesicles. Dishevelled accumulates on the dorsal side and inhibits GSK3. β-catenin can therefore accumulate on the dorsal side and, together with XTCF-3, induce the expression of siamois, which regulates down-stream dorsal development.
As such, the Wnt signaling system is one of only a limited number of signaling systems used during embryonic development to pattern the ultimate resultant morphological physical body construction plan. Clearly, Wnt signaling is triggered at several discrete time points during development, both at different developmental stages and within different tissues (see Table below).
TABLE 1GeneExpressionFunctionXWnt-1anterior neuralmid-/hindbrain boundaryXWnt-2 (=XWnt-2B)neural and heartnot knownXWnt-3-Aposterior neuralneural anteroposteriorpatterningXWnt-4neural, kidneykidney morphogenesis(pronephros)XWnt-5Aectodermnot knownXWnt-8ventral mesodermmesodermal patterningXWnt-8bforebrainnot knownXWnt-11dorsal marginal zonegastrulation movements
Early Xenopus development provides an excellent model system for studying the general questions of tissue-specific response to Wnt signaling. Before the onset of zygotic transcription at the Mid-Blastula Transition (MBT) phase, the Wnt pathway functions to establish the dorsal body axis. Only an hour or two later, after MBT, XWnt-8 functions to promote ventral and lateral mesoderm. These strict stage-specific responses to Wnt signaling could conceivably be induced by differential use of the canonical and alternative Wnt signal transduction pathways.
It is further known to those of skill in the art that Wnt genes are active in osteoblast cells. Wnt regulates bone deposition in embryos and in mature individuals. It has been found that Wnt signals impact the dorsal-ventral pattern in early Xenopus embryo. In late embryos, Wnt causes anterior-posterior patterning of the neural tissue, neural crest formation, and organogenesis. As such, it is desired to have compositions and methods for controlling Wnt signaling. Such compositions and methods would have impact on embryonic developmental processes such as anterior-posterior patterning and on bone deposition.
Background of Sost.
Sost is believed to be a Bone Morphogenic Protein (BMP) antagonist. Mutations in the human Sost gene on human chromosome 17 can result in sclerosteosis, which is an autosomal recessive sclerosing bone dysplasia characterized by progressive skeletal overgrowth. A high incidence of the bone dysplasia disorder, occurring as a result of a founder effect in affected individuals has been reported in the Afrikaner population of South Africa, where a majority of individuals are affected by the disorder. Homozygosity mapping in Afrikaner families, along with analysis of historical recombinants, localized sclerosteosis to an interval of ˜2 cm. between the loci D17S1787 and D17S930 on chromosome 17q12-q21. Affected Afrikaners carry a nonsense mutation near the amino terminus of the encoded protein, whereas an unrelated affected person of Senegalese origin carries a splicing mutation within the single intron of the gene. The Sost gene encodes a protein that shares structural and functional similarity with a class of cysteine knot-containing factors, including dan, cerberus, gremlin, and caronte. The specific and progressive effect on bone formation observed in individuals affected with sclerosteosis suggests that the Sost gene encodes a regulator of bone homeostasis.
As such, evidence is provided herein that the deficiency of the Sost gene product, a novel secreted protein expressed in osteoblasts, leads to the increased bone density in sclerosteosis. The two nonsense mutations, and the splice site mutation, are loss-of-function mutations. Previously, the precise function and working of Sost was believed unknown, an inhibitory effect on bone formation can be proposed since pathophysiological analysis indicated that sclerosteosis is primarily a disorder of increased formation of normal bone. While it is known that Sost impacts bone formation, it is desired herein to better delineate the mechanism of action and pathway of Sost's bone deposition activity. Previously, it has been hypothesized that Sost affected BMP rather than the Wnt pathway. Previous to our described invention herein, it was not known that Sost reacted with Wnt pathway elements. The Sost-Wnt pathway interaction can be alternatively direct or indirect in nature.
Background of LRP6.
LRP genes encode the low-density lipoprotein (LDL)-receptor-related proteins, LRP5 and LRP6. Human LRP5 and LRP6 share 71% amino-acid identity, and together with Arrow, form a distinct subgroup of the LRP family. Arrow, LRP5, and LRP6 each contain an extracellular domain with epidermal growth factor (EGF) repeats and low-density lipoprotein receptor (LDLR) repeats, followed by a transmembrane region and a cytoplasmic domain lacking recognizable catalytic motifs. An LRP6 mutation in mice results in pleiotropic defects recapitulating some, but not all, of the Wnt mutant phenotype. LRP5/LRP6 involvement in Wnt signaling and LRP function in Wnt-induced axis Xenopus embryos have been previously studied.
LRPs and Arrow in Drosophila are long single-pass transmembrane proteins. These proteins are of interest because they interact with and affect Wnt signaling. Arrow is genetically required for Wingless (Wg) signaling (Webril, 2000) and mouse LRP mutations are similar in phenotype to Wnt mutants (Pinson, 2000). In Xenopus, over-expression of LRP can activate Wnt signaling (Tamai, 2000). There is evidence that Wnts can bind directly to the extra-cellular domain of LRP and form a ternary complex with the Frizzled receptor (Tamai, 2000). Also, the cytoplasmic domain of LRP can interact with Axin (Mao, 2001). Thus, LRP/Arrow appear to be important to understanding Wnt.
As stated, the Frizzled (Fz) family of serpentine receptors function as Wnt receptors, but how Fz proteins transduce signaling is not understood. In Drosophila, Arrow phenocopies the Wingless (DWnt-1) phenotype, and encodes a transmembrane protein that is homologous to two members of the mammalian low-density lipoprotein receptor (LDLR)-related protein (LRP) family, LRP5 and LRP6. It is reported that'LRP6 functions as a co-receptor for Wnt signal transduction. In Xenopus embryos, LRP6 activated Wnt-Fz signaling, and induced Wnt responsive genes, dorsal axis duplication, and neural crest formation. An LRP6 mutant lacking the carboxyl intracellular domain blocked signaling by Wnt or Wnt-Fz, but not by Dishevelled or β-catenin, and inhibited neural crest development. The extracellular domain of LRP6 bound Wnt-1 and associated with Fz in a Wnt-dependent manner. This indicates that LRP6 is likely to be a component of the Wnt receptor complex.
Further, Wnt/β-catenin signaling induces dorsal axis formation through activation of immediate, early responsive genes, including nodal-related 3 (Xnr3) and Siamois (Sia). It has been shown that in two developmental processes dependent on the Wnt pathway in Xenopus—secondary axis and neural crest formation—LRP6 activates, but a dominant-negative LRP6 inhibits, Wnt signaling, providing compelling evidence that LRP6 is critical in Wnt signal transduction. LRP6 functions upstream of Dsh in Wnt-responding cells, synergizes with either Wnt or Fz, and importantly, is able to bind Wnt-1 and to associate with Fz in a Wnt-dependent manner. The simplest interpretation of these findings is that LRP6 is a component of the Wnt-Fz receptor complex.
Genetic studies of Arrow in Drosophila and LRP6 in mice strongly support this hypothesis. Data also indicates the possibility that Wnt-induced formation of the Fz-LRP6 complex assembles LRP6, Fz and their associated proteins, thereby initiating cytoplasmic signaling. Consistent with this notion, Wnt signal transduction requires intracellular regions of both Fz and LRP6, which harbor candidate protein-protein interaction motifs. Notably, Arrow does not exhibit Fz planar polarity phenotype, implying that Arrow-LRP6 may specify Wnt-Fz signaling towards the β-catenin pathway. How Fz, LRP6, and proteoglycan molecules, such as Dally, interact to mediate Wnt recognition/specificity, and signal transduction remains to be elucidated. Thus, it is understood that LRP interacts with Wnt. The present invention is designed and characterized to control LRP binding to Wnt and Fz, and, more particularly, to control LRP upstream.
Background of LRP5.
In humans, low peak bone mass is a recognized significant risk factor for osteoporosis. It has been reported that LRP5, encoding the LDLR-related protein 5, affects bone mass accrual during growth. Mutations in LRP5 cause the autosomal recessive disorder osteoporosis-pseudoglioma syndrome (OPPG). OPPG is an autosomal recessive disease, characterized by severe osteoporosis due to decreased bone formation and pseudoglioma resulting from failed regression of primary vitreal vasculature. Y. Gong, et al. (2001). Gain of gene function leads to high bone mass (HBM) phenotype as an autosomal dominant trait, whereas loss of function leads to osteoporosis.
It has been found that OPPG carriers have reduced bone mass when compared to age- and gender-matched controls. LRP5 expression by osteoblasts in situ has been demonstrated and LRP-5 has been shown to reduce bone thickness in mouse calvarial explant cultures. These data indicate that Wnt-mediated signaling via LRP5 affects bone accrual during growth and is important for the establishment of peak bone mass.
In mice, it has been found that LRP5 participates in bone formation and bone mass. Null mutation of LRP5 causes post-natal bone loss, resulting from decreased bone formation and osteoblast proliferation, independent of Runx2. M. Kato, et al. (2002). In contrast, transgenic mice expressing LRP5 with the HBM mutation G171V exhibit increased bone formation and bone mass, without skeletal developmental abnormalities. F. Bex, et al. (2002).
LRP5 appears to interact with the Wnt pathway since LRP5 with the HBM mutation prevents inhibition of Wnt signaling by Dikkopf-1. L. M. Boyden (2002); A. M. Zorn (2001). There is murine hybridization and microarray evidence that indicates Wnt signaling is involved in bone fracture repair. M. Hadjiargyrou (2002). Six additional mutations in LRP5, located in the amino-terminal domain near G171, have been identified. These mutations cause increased bone density, particularly in cortical bone. L. Van Wesenbeeck (2003).
Background of β-catenin.
β-catenin reports demonstrate its accumulation opposite the sperm entry point by the end of the first cell cycle. β-catenin continues to accumulate in dorsal (i.e., opposite the sperm entry point) but not ventral cytoplasm through the early cleavage stages. By the 16- to 32-cell stages, it accumulates in dorsal but not ventral nuclei. Remarkably, the pattern of dorsal accumulation of β-catenin closely parallels the ability of transplanted dorsal cells to induce an axis when implanted into host embryos. Thus, β-catenin is the first signaling molecule to show a dorso-ventral polarity in the early embryo. Combined with the loss-of-function data from Heasman et al., it is now clear that when fertilization elicits a cortical rotation, and displacement of material and organelles to the future dorsal side, it leads to a dorso-ventral asymmetry in β-catenin, which is required for axis formation.
Brannon et al. show that the HMG Box factor XTCF-3 directly binds the siamois promoter. In the absence of β-catenin, XTCF-3 inhibits gene expression. However, on the dorsal side of the embryo, β-catenin binds the XTCF-3, and, thus, activates the gene. This is notable because siamois is a homeobox gene likely playing a major role in specifying formation of Spemann's Organizer. Therefore, a dorso-ventral difference in β-catenin forms within an hour or two of fertilization, directly regulating a key homeobox gene in the blastula, thus contributing to formation of Spemann's Organizer on the dorsal side of the gastrula.
β-catenin not only impacts development, but it influences bone development in adults. Regulation of osteoblasts results from accumulation of β-catenin in the cell. It is desired to have methods and compositions for controlling bone deposition. It is known that the Wnt pathway controls accumulation of β-catenin, which regulates osteoblast expression. It is desired to control and inhibit osteoblast regulation by preventing Wnt pathway activation. For this reason, the present invention includes nucleic acid molecules and amino acid sequences for controlling Wnt.